Nucleolin promotes Ang II-induced phenotypic transformation of vascular smooth muscle cells by regulating EGF and PDGF-BB.

RNA-binding properties of nucleolin play a fundamental role in regulating cell growth and proliferation. We have previously shown that nucleolin plays an important regulatory role in the phenotypic transformation of vascular smooth muscle cells (VSMCs) induced by angiotensin II (Ang II). In the present study, we aimed to investigate the molecular mechanism of nucleolin-mediated phenotypic transformation of VSMCs induced by Ang II. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) inhibitors were used to observe the effect of Ang II on phenotypic transformation of VSMCs. The regulatory role of nucleolin in the phenotypic transformation of VSMCs was identified by nucleolin gene mutation, gene overexpression and RNA interference technology. Moreover, we elucidated the molecular mechanism underlying the regulatory effect of nucleolin on phenotypic transformation of VSMCs. EGF and PDGF-BB played an important role in the phenotypic transformation of VSMCs induced by Ang II. Nucleolin exerted a positive regulatory effect on the expression and secretion of EGF and PDGF-BB. In addition, nucleolin could bind to the 5' untranslated region (UTR) of EGF and PDGF-BB mRNA, and such binding up-regulated the stability and expression of EGF and PDGF-BB mRNA, promoting Ang II-induced phenotypic transformation of VSMCs.

A minimally invasive approach to induce myocardial infarction in mice without thoracotomy.

Acute myocardial infarction (MI) is a leading cause of morbidity and mortality in the world. Traditional method to induce MI by left coronary artery (LCA) ligation is typically performed by an invasive approach that requires ventilation and thoracotomy, causing serious injuries in animals undergoing this surgery. We attempted to develop a minimally invasive method (MIM) to induce MI in mice. Under the guide of ultrasound, LCA ligation was performed in mice without ventilation and chest-opening. Compared to sham mice, MIM induced MI in mice as determined by triphenyltetrazolium chloride staining and Masson staining. Mice with MIM surgery revealed the reductions of LVEF, LVFS, E/A and ascending aorta (AAO) blood flow, and the elevations of S-T segment and serum cTn-I levels at 24 post-operative hours. The effects of MI induced by MIM were comparable to the effects of MI produced by traditional method in mice. Importantly, MIM increased the survival rates and caused less inflammation after the surgery of LCA ligation, compared to the surgery of traditional method. Further, MIM induced angiogenesis and apoptosis in ischaemic hearts from mice at postoperative 28 days as similarly as traditional method did. Finally, the MIM model was able to develop into the myocardial ischaemia/reperfusion model by using a balloon catheter with minor modifications. The MI model is able to be efficiently induced by a minimally invasive approach in mice without ventilation and chest-opening. This new model is potentially to be used in studying ischaemia-related heart diseases.

MiR-15b-5p Regulates Collateral Artery Formation by Targeting AKT3 (Protein Kinase B-3).

OBJECTIVE: To identify circulating microRNAs that are differentially expressed in severe coronary heart disease with well or poorly developed collateral arteries and to investigate their mechanisms of action in vivo and in vitro. APPROACH AND RESULTS: In our study, we identified a circulating microRNA, miR-15b-5p, with low expression that, nevertheless, characterized patients with sufficient coronary collateral artery function. Moreover, in murine hindlimb ischemia model, in situ hybridization identified that miR-15b-5p was specifically expressed in vascular endothelial cells of adductors in sham group and was remarkably downregulated after femoral artery ligation. Overexpressed miR-15b-5p significantly inhibited arteriogenesis and angiogenesis in mice. In vitro, both under basal and vascular endothelial growth factor stimulation, loss-of-function or gain-of-function studies suggested that miR-15b-5p significantly promoted or depressed the migration and proliferation of endothelial cells. We identified AKT3 (protein kinase B-3) as a direct target of miR-15b-5p. Interestingly, AKT3 deficiency by injection with Chol-AKT3-siRNA obviously suppressed arteriogenesis and the recovery of blood perfusion after femoral ligation in mice. CONCLUSIONS: These results indicate that circulating miR-15b-5p is a suitable biomarker for discriminating between patients with well-developed or poorly developed collaterals. Moreover, miR-15b-5p is a key regulator of arteriogenesis and angiogenesis, which may represent a potential therapeutic target for ischemic disease.

Asymmetric dimethylarginine triggers macrophage apoptosis via the endoplasmic reticulum stress pathway.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is emerging as a key contributing factor in atherogenesis, a process in turn known to involve macrophage apoptosis. The aim of this study was to determine the effect of ADMA on macrophage apoptosis, with specific reference to the endoplasmic reticulum (ER) stress pathway. Macrophage apoptosis was evaluated by Annexin V- Propidium iodide (PI) and Hoechst 33258 staining assays. Levels of the ER stress marker glucose regulated protein 78 (GRP78) were characterized by western blot. Levels of the proapoptotic C/EBP-homologous protein (CHOP) were evaluated by western blot and reverse transcription polymerase chain reaction (RT-PCR), and caspase-4 activity was measured using a colorimetric protease assay kit. We observed ADMA dose- and time-dependent increases in macrophage levels of GRP78. Similar ADMA dose- and time-dependent increases were detected in intracellular caspase-4 activity and macrophage apoptosis, all of which were sensitive to treatment with siRNAs for protein kinase RNA-like ER kinase and inositol-requiring protein-1 (IRE1), the ADMA antagonist L-arginine, as well as inhibitors of eukaryotic translation initiation factor-2 (salubrinal), IRE1 (irestatin 9389), and c-Jun N-terminal kinase (SP600125). Our results indicate that ADMA triggers macrophage apoptosis via the ER stress pathway.

Nicorandil alleviates cardiac remodeling and dysfunction post -infarction by up-regulating the nucleolin/autophagy axis.

OBJECTIVE: The present study aimed to investigate whether the drug nicorandil can improve cardiac remodeling after myocardial infarction (MI) and the underlying mechanisms. METHODS: Mouse MI was established by the ligation of the left anterior descending coronary artery and H9C2 cells were cultured to investigate the underlying molecular mechanisms. The degree of myocardial collagen (Col) deposition was evaluated by Masson's staining. The expressions of nucleolin, autophagy and myocardial remodeling-associated genes were measured by Western blotting, qPCR, and immunofluorescence. The apoptosis of myocardial tissue cells and H9C2 cells were detected by TUNEL staining and flow cytometry, respectively. Autophagosomes were observed by transmission electron microscopy. RESULTS: Treatment with nicorandil mitigated left ventricular enlargement, improved the capacity of myocardial diastolic-contractility, decreased cardiomyocyte apoptosis, and inhibited myocardial fibrosis development post-MI. Nicorandil up-regulated the expression of nucleolin, promoted autophagic flux, and decreased the expressions of TGF-ß1 and phosphorylated Smad2/3, while enhanced the expression of BMP-7 and phosphorylated Smad1 in myocardium. Nicorandil decreased apoptosis and promoted autophagic flux in H2O2-treated H9C2 cells. Autophagy inhibitors 3-methyladenine (3MA) and chloroquine diphosphate salt (CDS) alleviated the effects of nicorandil on apoptosis. Knockdown of nucleolin decreased the effects of nicorandil on apoptosis and nicorandil-promoted autophagic flux of cardiomyocytes treated with H2O2. CONCLUSIONS: Treatment with nicorandil alleviated myocardial remodeling post-MI through up-regulating the expression of nucleolin, and subsequently promoting autophagy, followed by regulating TGF-ß/Smad signaling pathway.

Nucleolin Mediates LPS-induced Expression of Inflammatory Mediators and Activation of Signaling Pathways.

In this study, we investigated the effects of nucleolin on lipopolysaccharide (LPS)-induced activation of MAPK and NF-KappaB (NF-κB) signaling pathways and secretion of TNF-α, IL-1ß and HMGB1 in THP-1 monocytes. Immunofluorescence assay and Western blotting were used to identify the nucleolin expression in cell membrane, cytoplasm and nucleus of THP-1 monocytes. Inactivation of nucleolin was induced by neutralizing antibody against nucleolin. THP-1 monocytes were pretreated with anti-nucleolin antibody for 1 h prior to LPS challenge. The irrelevant IgG group was used as control. Secretion of inflammatory mediators (TNF-α, IL-1ß and HMGB1) and activation of MAPK and NF-κB/I-κB signaling pathways were examined to assess the effects of nucleolin on LPS-mediated inflammatory response. Nucleolin existed in cell membrane, cytoplasm and nucleus of THP-1 monocytes. Pretreatment of anti-nucleolin antibody significantly inhibited the LPS-induced secretion of TNF-α, IL-1ß and HMGB1. P38, JNK, ERK and NF-κB subunit p65 inhibitors could significantly inhibit the secretion of IL-1ß, TNF-α and HMGB1 induced by LPS. Moreover, the phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65) was significantly increased after LPS challenge. In contrast, pretreatment of anti-nucleolin antibody could significantly inhibit the LPS-induced phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65). However, the irrelevant IgG, as a negative control, had no effect on LPS-induced secretion of TNF-α and IL-1ß and phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65). We demonstrated that nucleolin mediated the LPS-induced activation of MAPK and NF-κB signaling pathways, and regulated the secretion of inflammatory mediators (TNF-α, IL-1ß and HMGB1).

Nucleolin promotes Ang II­induced phenotypic transformation of vascular smooth muscle cells via interaction with tropoelastin mRNA.

The current study aimed to clarify the role of nucleolin in the phenotypic transformation of vascular smooth muscle cells (VSMCs) and to preliminarily explore its underlying mechanism. The spatial and temporal expression patterns of nucleolin, and the effects of angiotensin II (Ang II) on the expression of VSMC phenotypic transformation markers, α­smooth muscle­actin, calponin, smooth muscle protein 22α and osteopontin were investigated. The effects of nucleolin on VSMC phenotypic transformation and the expression of phenotypic transformation­associated genes, tropoelastin, epiregulin and fibroblast growth factor 2 (b­FGF), were determined. Protein­RNA co­immunoprecipitation was used to investigate the potential target genes regulated by the nucleolin in phenotypic transformation of VSMCs. Finally, the stability of tropoelastin mRNA and the effects of nucleolin on the expression of tropoelastin were assayed. The results revealed that Ang II significantly promoted the phenotypic transformation of VSMCs. The expression of nucleolin was gradually upregulated in VSMCs treated with Ang II at different concentrations for various durations. Ang II induced nucleolin translocation from the nucleus to cytoplasm. Additionally, Ang II significantly promoted the phenotypic transformation of VSMCs. Overexpression and silencing of nucleolin regulated the expressions of tropoelastin, epiregulin and b­FGF. There was an interaction between tropoelastin mRNA and nucleolin protein, promoting the stability of tropoelastin mRNA and enhancing the expression of tropoelastin at the protein level. Upregulation of nucleolin had an important role in Ang II­induced VSMC phenotypic transformation, and its underlying mechanism may be through interacting with tropoelastin mRNA, leading to its increased stability and protein expression. The findings provide a new perspective into the regulatory mechanism of VSMC phenotypic transformation.

[Role of cell-surface nucleolin in lipopolysaccharide-stimulated expression and secretion of TNF-alpha and IL-1beta].

OBJECTIVE: To explore the role of cell-surface nucleolin in lipopolysaccharide (LPS)-stimulated expression and secretion of TNF-alpha and IL-1beta in human THP-1 monocytes. METHODS: Immuno-fluorescence assay and Western blot were used to identify the expression of nucleolin on the surface of THP-1 monocytes. Inactivation of nucleolin was induced by anti-nucleolin monoclonal antibody blockage, and the expression and secretion of TNF-alpha and IL-1beta were observed by using reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immuno-sorbent assay (ELISA)respectively in LPS-mediated human THP-1 monocyte inflammatory model. RESULTS: Immuno-fluorescence showed that nucleolin was localized on the cell surface of THP-1 monocytes as indicated by dotted red fluorescence. Western blot assay indicated that nucleolin existed in the cell membrane fractions. RT-PCR assay showed that the expressions of TNF-alpha and IL-1beta mRNA significantly increased at 2 h and 3 h after the treatment with 1000 microg/L LPS. After 1 h pretreatment with anti-nucleolin antibody, the levels of TNF-alpha and IL-1beta mRNA decreased compared with an anti-nucleolin antibody untreated group and an irrelevant IgG+LPS group (P<0.05). ELISA assay showed that the pretreatment with anti-nucleolin antibody inhibited significantly the secretion of LPS-induced levels of TNF-alpha and IL-1beta after 4, 12 and 24 h treatment with 1000 microg/L LPS. CONCLUSION: Nucleolin expresses on the cell surface of THP-1 monocytes and involves in the LPS-mediated expression and secretion of TNF-alpha and IL-1beta.

Hypoxia-elicited mesenchymal stem cell-derived exosomes facilitates cardiac repair through miR-125b-mediated prevention of cell death in myocardial infarction.

Exosomes (Exo) secreted from hypoxia-conditioned bone marrow mesenchymal stem cells (BM-MSCs) were found to be protective for ischemic disease. However, the role of exosomal miRNA in the protective effect of hypoxia-conditioned BM-MSCs-derived Exo (Hypo-Exo) remains largely uncharacterized and the poor specificity of tissue targeting of Exo limits their clinical applications. Therefore, the objective of this study was to examine the effect of miRNA in Hypo-Exo on the repair of ischemic myocardium and its underlying mechanisms. We further developed modified Hypo-Exo with high specificity to the myocardium and evaluate its therapeutic effects. Methods: Murine BM-MSCs were subjected to hypoxia or normoxia culture and Exo were subsequently collected. Hypo-Exo or normoxia-conditioned BM-MSC-derived Exo (Nor-Exo) were administered to mice with permanent condition of myocardial infarction (MI). After 28 days, to evaluate the therapeutic effects of Hypo-Exo, infarction area and cardio output in Hypo-Exo and Nor-Exo treated MI mice were compared through Masson's trichrome staining and echocardiography respectively. We utilized the miRNA array to identify the significantly differentially expressed miRNAs between Nor-Exo and Hypo-Exo. One of the most enriched miRNA in Hypo-Exo was knockdown by applying antimiR in Hypoxia-conditioned BM-MSCs. Then we performed intramyocardial injection of candidate miRNA-knockdown-Hypo-Exo in a murine MI model, changes in the candidate miRNA's targets expression of cardiomyocytes and the cardiac function were characterized. We conjugated Hypo-Exo with an ischemic myocardium-targeted (IMT) peptide by bio-orthogonal chemistry, and tested its targeting specificity and therapeutic efficiency via systemic administration in the MI mice. Results: The miRNA array revealed significant enrichment of miR-125b-5p in Hypo-Exo compared with Nor-Exo. Administration of miR-125b knockdown Hypo-Exo significantly increased the infarction area and suppressed cardiomyocyte survival post-MI. Mechanistically, miR-125b knockdown Hypo-Exo lost the capability to suppress the expression of the proapoptotic genes p53 and BAK1 in cardiomyocytes. Intravenous administration of IMT-conjugated Hypo-Exo (IMT-Exo) showed specific targeting to the ischemic lesions in the injured heart and exerted a marked cardioprotective function post-MI. Conclusion: Our results illustrate a new mechanism by which Hypo-Exo-derived miR125b-5p facilitates ischemic cardiac repair by ameliorating cardiomyocyte apoptosis. Furthermore, our IMT- Exo may serve as a novel drug carrier that enhances the specificity of drug delivery for ischemic disease.

[Endogenous myocardial protection: present questions and prospects].

Since the findings of Murry and Currie et al. that ischemic preconditioning (IPC) and heat shock response (HSR) could protect evidently myocardium against ischemia-reperfusion injury in the middle of 1980s, endogenous myocardial protection has drawn widespread attentions. A great quantity of studies completed during the past 25 years made much progress in endogenous myocardial protection. Abundant research experiences have been accumulated and a basic theoretical framework has been established in this field. However, there are still many questions need to be solved. In this review, we focused on clarifying some hot questions and important future directions in IPC, heat shock proteins (HSPs), research models and strategies in endogenous myocardial protection.

[Effect of heat shock protein 47 on the expression of collagen I induced by TGF-beta(1) in hepatic stellate cell-T6 cells].

OBJECTIVE: To determine the effect of heat shock protein 47 (HSP47) on the expression of collagen I induced by transforming growth factor beta(1) (TGF-beta(1)) in hepatic stellate cell-T6 (HSC-T6) cells. METHODS: We used 1 ng/mL and 10 ng/mL recombinant human TGF-beta(1) to stimulate the cultured HSC-T6 cells. Heat shock response (HSR) and antisense oligonucleotides of HSP47 were used to induce and block the expression of HSP47, respectively. The expressions of HSP47 and collagen I were detected by Western blot and the cell viability was observed by MTT assay. RESULTS: Both HSP47 and collagen I were expressed in normal HSC-T6 cells. Collagen I and HSP47 expression could be induced by both 1 ng/mL and 10 ng/mL TGF-beta(1) and collagen I was expressed the most after the treatment with 10 ng/mL TGF-beta(1). Although HSR could not affect the synthesis of collagen I as it induced the HSP47 expression, HSR could promote the expression of collagen I induced by TGF-beta(1). With no effect on the cell viability, antisense oligonucleotides could significantly inhibit HSR-mediated HSP47 expression and TGF-beta(1)-induced collagen I synthesis. CONCLUSION: Over-expression of HSP47 enhances TGF-beta(1)-induced expression of collagen I in HSC-T6 cells, and HSP47 may play important roles in the process of hepatic fibrosis.

[HSF1 inhibits heat stress-induced apoptosis in Raw264.7 macrophages].

OBJECTIVE: To observe the effect of heat shock factor 1 (HSF1) on heat stress-induced apoptosis in Raw264.7 macrophages. METHODS: Raw264.7 cells transfected with pcDNA3.1 and pcDNA3.1-HSF1 were exposed to heat stress (42.5 degrees C +/- 0.5 degrees C) for 1 h and recovered at 37 degrees C for 6, 9, 12, and 24 h respectively. Flow cytometry (FCM), Hoechst 33258 staining and DNA ladder assays were performed to assess the apoptosis. RESULTS: After heat stress, FCM showed that apoptotic cells were increased significantly and reached the peak at 9 h in Raw 264.7 cells transfected with pcDNA3.1, and were characterized with classical morphologic changes including apoptotic body and nuclear condensation. Agarose gel electrophoresis showed that "DNA ladder" could be observed clearly at 6, 9, and 12 h after the heat stress. But the overexpression of HSF1 could reduce the number of apoptotic cells and inhibit DNA fragmentation. CONCLUSION: HSF1 can inhibit heat stress-induced apoptosis in Raw264.7 macrophages.

[Immortalization of embryonic fibroblasts in heat shock transcription factor 1 knockout mouse].

OBJECTIVE: To establish immortalized embryonic fibroblast lines in heat shock transcription factor 1 (HSF1) HSF1-/- and HSF1+/+ mice and to provide experimental models to study the function of HSF1. METHODS: A mammalian expression vector (pSV3neo) containing the SV40 large T antigen was used to transfect the HSF1-/- and HSF1+/+ mouse embryonic fibroblast using Lipofectamine 2000. Colonies were screened by G418 and expanded to immortalized cell lines. PCR was used to detect the integration of the large T antigen with genome in the mouse embryonic fibroblast. Expression of SV40 large T antigen gene in expanded cells was identified by RT-PCR. HSP70 expression was examined by Western blot in the embryonic fibroblast lines. RESULTS: The stable growth and serial propagation were observed in the HSF1-/- and HSF1+/+ cell lines for six months. The mRNA of SV40 T antigen gene expressed in the two cell lines. HSP70 expression could not be induced in the heat-treated HSF1-/- mouse embryo fibroblasts. CONCLUSION: The immortalized cells of HSF1+/+ and HSF1-/- mouse embryo fibroblasts are successfully established.

[Oxidative stress-induced accumulation of heat shock protein 70 within nucleolus].

OBJECTIVE: To investigate the effect of oxidative stress on the accumulation of heat shock protein 70 (HSP70) within C2C12 myogenic cells. METHODS: Heat shock response (42 degrees C for 1 h and recovery for 12 h at 37 degrees C) was used to induce the expression of heat shock protein 70. We constructed a recombinant plasmid of HSP70 with enhanced green fluorescent protein (EGFP). After being transfected transiently into C2C12 cells, immunoblotting was used to detect the expression of HSP70 induced by heat shock response and transfection. Immunocytochemistry, fluorescent microscopy and immunoblotting were used to detect the translocation of HSP70. RESULTS: Immunoblotting showed that the overexpression of HSP70 was induced by heat shock response and transient transfenction. HSP70 localized within the cytoplasm of the normal cells, but HSP70 translocated from the cytoplasm to the nucleus and the nucleolus at 1 h after the treatment of oxidative stress (0.5 mmol/L H2O2) by using immunocytochemistry, fluorescent microscopy and immunoblotting for cellular partial proteins. CONCLUSION: Oxidative stress may induce the accumulation of heat shock protein 70 within the nucleolus.

[Characteristics and expression of Mip5, a novel gene associated with myocardial ischemia/reperfusion in rats].

OBJECTIVE: To determine the characteristics of a novel gene Mip5 (GenBank accession number AY553870) and its expression under physiological and pathological conditions. METHODS: The characteristics of Mip5 were analyzed by bioinformatic programs including BLAST, spidey, psort, ClustalW and so on. RT-PCR was performed to detect Mip5 expression. RESULTS: Bioinformatic analysis showed that Mip5 gene lied in the 13th chromosome and contained 8 exons and 7 introns, its open reading frame contained 909 bp and its protein production was 302 amino acid residues including 6 kelth domains. Under normal conditions, MIP5 expressed abundantly in the heart, brain and kidney, but its expression could not be detected in the liver and muscle. Expression of Mip5 gene was increased significantly after ischemia-reperfusion compared with the sham groups, and reached its peak at 3 h and recovered at 12 h after the reperfusion. Conclusion Mip5 gene is a novel gene containing a putative open reading frame of 302 amino acids residues and may play an important role in rat cardiomyocytes suffering ischemia processing.

[Effect of nucleolin down-regulation on the proliferation and apoptosis in C2C12 cells].

OBJECTIVE: To clarify the effect of nucleolin on the proliferation and apoptosis in C2C12 cells. METHODS: After inhibiting the expression of nucleolin using antisense oligonucleotides, the cellular proliferation was determined by MTT, and the apoptosis was detected by flow cytometry (FCM) assays and DNA ladder assays. RESULTS: After being transfected with antisense oligonucleotides for 24 hours, Western blotting showed that the expression of nucleolin was repressed significantly. In cells treated with antisense oligonucleotides, the cellular proliferation was obviously inhibited; the apoptotic cell increased significantly; and the "DNA ladder" was clearly observed. But the sense and random oligonucleotides had no effect on the cellular proliferation and apoptosis. CONCLUSION: The down-regulation of nucleolin can inhibit the cellular proliferation and initiate the apoptosis in C2C12cells.

[Effect of heat shock response on the cleavage of nucleolin induced by oxidative stress].

OBJECTIVE: To observe the cleavage of nucleolin (C23) during apoptosis induced by oxidative stress and to clarify the effect of heat shock response (HSR) on the cleavage of nucleolin and its possible molecular mechanism. METHODS: We added 0.5 mmol/L peroxide hydrogen (H2O2 ) into cultured cells to mimic oxidative stress. Apoptosis and cleavage of C23 were detected using caspase-3 colorimetric assay and Western blotting respectively. HSR was performed to observe the effect of HSR on cleavage of C23 induced by oxidative stress, and over-expressions of HSP70 and HSP25 were detected by Western blotting. RESULTS: Activity of caspase-3 increased significantly after 2 hours of 0.5 mmol/L H2O2 treatment, and reached the peak after 12 hours. The cleavage of C23 appeared 30 minutes to 1 hour after the treatment of H2O2 as indicated by a cleaved fragmentation of 80 kD, which was significantly inhibited by HSR. Moreover, HSR could induce HSP70 and HSP25 over-expressions. CONCLUSION: Oxidative stress can induce the activation of caspase-3, cleavage of C23, and apoptosis. HSR can significantly inhibit the cleavage of C23 induced by oxidative stress, which is related to the over-expressions of HSP70, HSP25, and other stress proteins.

Ischemic postconditioning protects cardiomyocytes against ischemia/reperfusion injury by inducing MIP2.

Cardiomyocytes can resist ischemia/reperfusion (I/R) injury through ischemic postconditioning (IPoC) which is repetitive ischemia induced during the onset of reperfusion. Myocardial ischemic preconditioning up-regulated protein 2 (MIP2) is a member of the WD-40 family proteins, we previously showed that MIP2 was up-regulated during ischemic preconditioning (IPC). As IPC and IPoC engaged similar molecular mechanisms in cardioprotection, this study aimed to elucidate whether MIP2 was up-regulated during IPoC and contributed to IPoC-mediated protection against I/R injury. The experiment was conducted on two models, an in vivo open chest rat coronary artery occlusion model and an in vitro model with H9c2 myogenic cells. In both models, 3 groups were constituted and randomly designated as the sham, I/R and IPoC/hypoxia postconditioning (HPoC) groups. In the IPoC group, after 45 min of ischemia, hearts were allowed three cycles of reperfusion/ischemia phases (each of 30 s duration) followed by reperfusion. In the HPoC group, after 6 h of hypoxia, H9c2 cells were subjected to three cycles of 10 minute reoxygenation and 10 minute hypoxia followed by reoxygenation. IPoC significantly reduced the infarct size, plasma level of Lactate dehydrogenase and creatine kinase MB in rats. 12 h after the reperfusion, MIP2 mRNA levels in the IPoC group were 10 folds that of the sham group and 1.4 folds that of the I/R group. Increased expression of MIP2 mRNA and attenuation of apoptosis were similarly observed in the HPoC group in the in vitro model. These effects were blunted by transfection with MIP2 siRNA in the H9c2 cells. This study demonstrated that IPoC induced protection was associated with increased expression of MIP2. Both MIP2 overexpression and MIP2 suppression can influence the IPoC induced protection.